Maintenance of extracellular fluid volume homeostasis is essential for hemodynamic stability, and abnormalities of renal sodium handling have been linked to cardiovascular disease and hypertension. Ultimate regulation of sodium excretion occurs in the distal nephron via conductive transport through amiloride sensitive epithelial Na+ channel (ENaC). ENaC expression and activity in the apical membrane of epithelial cells is the rate limiting step in Na+ reabsorption not only in the kidney collecting duct, but in airway epithelia and colon as well. Aldosterone is a major regulator of ENaC expression and activity in responsive epithelia. Studies are designed to examine the mechanism of aldosterone regulation of ENaC. Work in the previous grant period has established that aldosterone stimulates the post-translational methylation of betaENaC which alters channel gating. Studies are now proposed to examine the site and mechanism of this regulation and potentially define a gating site in ENaC. We have demonstrated that EnaC appears to be localized, in part at least, to specialized areas of membrane called lipid rafts and this localization is regulated by aldosterone. Raft association of ENaC is a new finding and could be important for ENaC trafficking, apical membrane expression and association with regulatory proteins. We will examine the mechanism by which lipid raft association affects ENaC function and aldosterone regulation.